The culture medium potato dextrose agar (PDA) and Roswell Park Memorial Institute (RPMI) medium were purchased from Hi-Media Laboratory Mumbai, India. Rice (BPT 5204 sona masoori) was purchased from the local market for solid-state fermentation. The analytical-grade hexane and ethyl acetate were used for the extraction of pigments from fermented rice. The HPLC-grade acetonitrile was used for HPLC analysis. Tris (hydroxymethyl) amino-methane, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC), and 3-(N-morpholino) propanesulfonic acid (MOPS) were obtained from Sigma Chemicals St. Louis, MO, USA.

M. purpureus CFR410-11, a mutant, was developed earlier in the CSIR-CFTRI laboratory and used for pigment production (Dhale et al., 2007b; Dhale and Mohan-Kumari, 2014). The Penicillium expansum, MTCC 4900 Rhizopus stolinfer MTCC 10595, and Aspergillus niger MTCC 8652 were procured from Microbial-Type Culture Collection (MTCC), Chandigarh, India. The fungi were maintained on a PDA slant at 4°C by sub-culturing every 30 days. In total, 1-week-old slants were used for the preparation of spore suspension in 0.85% NaCl and Tween 20 (0.5%). To cultivate M. purpureus, the solid-state medium was prepared by autoclaving 10 g of rice in 20 ml of distilled water (1:2 w/v) for 20 min at 115°C. A volume of 1 ml of M. purpureus spore suspension (≈2 × 10⁵) was inoculated to rice medium and incubated for 10–12 days at 30°C (Hotcold S, J.P. Selecta, Barcelona, Spain). Intermittent shaking of the culture flasks (manually) allowed the uniform growth of M. purpureus. After fermentation, the red rice was dried at 40–45°C for 24 h.

The dried fermented rice was ground using an electric grinder. Approximately 10 g of fermented rice powder was used to extract the pigment in a semi-automatic solvent extractor (148 series, VELP Scientifica, Italy). The pigments were extracted sequentially twice in hexane, ethyl acetate, and ethanol. The pigment extracts were filtered through Whatman No. 1 filter paper and concentrated using a rotary flash evaporator (Buchi, Switzerland). After flash evaporation, the extract was lyophilized (Labconco, Kansas City) for further analysis.

The three different methods for radial growth, germination assay, minimum inhibitory concentration (MIC), and minimum fungicidal concentration (MFC) were used to determine the antifungal activity. The extracted M. purpureus pigments were dissolved appropriately in DMSO, PDA, or RPMI 1640 media for the assay conditions.

The M. purpureus pigment extracts (25 mg) mixed in PDA (130 ml) were sterilized. The aliquot of 20 ml of PDA was poured into the Petri plates and the control plates were maintained without any pigment extracts. These plates were inoculated by point inoculation or with 10 μl (≈1 × 10⁴) of fungal spore suspension on the PDA plates. The plates were incubated aerobically at 30°C. The radial growth (colony diameter) of mycelia on plates was measured after 4–5 days of incubation. The assays were carried out in triplicate. Each datum point is the mean for at least four measurements of a growing colony. The inhibitory activity of the M. purpureus pigment extracts was assayed based on the hyphal radial growth rates of fungi (Rossana et al., 2011). The experiments were carried out in triplicates and the data were expressed as the mean ± standard deviation. The percent radial growth inhibition was calculated from the mean values using the following formula:

The effect of M. purpureus pigment extracts on the germination of conidia was determined (Magnusson and Schnürer, 2001). The test fungi were grown on the PDA slant for 1 week. The spore suspension was prepared in sterile water containing 0.5% Tween 80. The spores suspension (10 μl) was added to the 500 μl of RPMI medium containing 75 μl of pigment extracts in centrifuge tubes. These tubes were kept at 30°C under gentle shaking, and the test samples were drawn at 8 and 24 h. The germ tube growth was observed under a microscope at 40X magnification.

Monascus pigment extracts were serially diluted in RPMI 1640 to determine the MIC (Rossana et al., 2011). The RPMI 1640 (150 μl) medium was dispensed in each well (flat bottom). The stock solution of pigment extract 25 mg/ml was prepared in DMSO. The pigment extracts, 150 μl, were added to a well and serially diluted (concentrations 150, 75, 37.5, 18.75, 9.38, 4.69, 2.34, and 1.17). To this, 10-μl spore suspension (≈1X10⁴) was inoculated in each well. The controls were maintained without samples to demonstrate the growth of fungal spores. While amphotericin-B (3 mg/ml) was used as a positive control. The plates were incubated at 30°C under mild stirring conditions and observed after 48 and 72 h of incubation. The MIC was defined as the lowest concentration of pigment extract, which inhibited visual fungal growth. All assays for antifungal activity were carried out at least in triplicate. The minimum concentration pigment that showed ≥99.9% reduction of the original inoculums was recorded as the MFC.

To determine whether the Monascus pigment extracts exert fungicidal activity against the test fungal mycelia (Ben-Ami et al., 2010), the viability staining with bis-(1,3- dibutylbarbituric acid) trimethine oxonol (DiBAC) on pigment treated and treated mycelia. The test fungal spores were grown to mycelia in the RPMI 1640 medium in micro-centrifuge tubes at 30°C with shaking for 36–48 h. After incubation, the tubes were centrifuged at 10,000 g for 10 min to remove the RPMI medium. The mycelia were resuspended in 500 μl RPMI containing 75 μl of Monascus pigment extracts. The amphotericin-B and medium without samples were the positive and negative controls, respectively. These tubes were incubated at 37°C for 6 h with gentle shaking. After incubation, the mycelia were washed twice in 3-(N-morpholino) propanesulfonic acid at pH 7 (MOPS7). The stain DiBAC prepared in 100% ethanol (1 mg/ml) was added to the tubes at the final concentration of 2 μg/ml in MOPS7. Tubes were incubated at room temperature in the dark for 1 h with gentle shaking (70–100 rpm). Again the mycelia were washed in MOPS7 and stored on ice until fluorescent microscopic observations. The images were captured under a triple-band fluorescent microscope (Olympus BX-51; Olympus, Melville, NY) using the fluorescein isothiocyanate (FITC) filter and bright field.

A thin-layer chromatography (TLC) analysis was conducted using a Silica gel 60 F254 TLC plate (Merck, Germany) with n-hexane/ethyl acetate/petroleum ether (30:17:5) as the developing solvent (Shi et al., 2017). Furthermore, the extracted pigments were subjected to high-performance liquid chromatography (HPLC). The extracted pigments were dissolved in the mixture of acetonitrile/water (70:30 v/v). The samples were filtered through a 0.45-μm nylon filter and subjected to HPLC (waters liquid chromatograph, PDA detector) in a C₁₈ analytical column a 250 × 4.6 mm i.d., 5 μm (Sigma, Discovery Suppleco, USA) carrying a UV–visible detector. Chromatographic separation was achieved by injecting 20 μl of the sample with isocratic elution of acetonitrile:water (70:30 v/v) at a flow rate of 1 mL min⁻¹. The elution was monitored at 495 nm for 45 min (Dhale et al., 2018). The pigment extracts were dissolved in respective solvents at the appropriate concentration to analyze the qualitative nature of the pigment. The UV–visible spectrum was recorded using a UV-1800 Shimadzu spectrophotometer at room temperature from 300 to 700 nm.

The radial growth inhibitory activities of the pigment extracts were tested against several fungi. The pigment extracts incorporated in the PDA medium have inhibited the radial growth of fungi (Figure 1). The growth inhibition was measured on PDA after 4–5 days of incubation at 30°C. The growth of tested fungi against the pigment was variously affected. The pigment extracted in hexane has shown the highest percentage inhibitory activity (48.28 ± 0.48%) against the A. niger MTCC 8652 compared with the pigment extracted in ethyl acetate and ethanol. While P. expansum MTCC 4900 (19.50 ± 2.0%) and R. stolinefer MTCC 10595 (22.02 ± 0.58%) were weakly inhibited by the pigment extracted in hexane. The comparative radial growth inhibition assay of pigment against tested fungi indicated that A. niger MTCC 8652 was more susceptible than P. expansum MTCC 4900 and R. stolinefer MTCC 10595 (Table 1). Whereas, all the pigment extracts have shown a similar growth inhibition of P. expansum MTCC 4900 and R. stolinefer MTCC 10595.

The M. purpureus pigment extracts have shown in vitro antifungal activity against P. expansum MTCC 4900, R. stolinfer MTCC 10595, and A. niger MTCC 8652. Susceptibility testing by broth micro-dilution in RPMI 1640 generally revealed clear endpoints with complete growth inhibition. The MIC of pigment extracted in hexane and ethanol for the tested fungi was 1563.3 μg/ml. The pigment extracted in ethyl acetate showed the MIC at a concentration of 780 μg/ml. The MIC of the standard drug amphotericin-B was 195 μg/ml. While the pigment extracts have shown a fungicidal effect at a concentration of two to three times the MIC. Furthermore, the micro broth dilution assay revealed the MFC at 3126.7 μg/ml (Table 2). In contrast, the amphotericin-B MFCs for all the fungi tested were 390 μg/ml.

After incubation for 8 h and 12 h at 30°C in the presence and absence of pigment extracts, the spores of P. expansum MTCC 4900, R. stolinfer MTCC 10595, and A. niger MTCC 8652 were observed under the microscope. The spores grown in the presence of pigment extracts and amphotericin-B did not show the formation of the germ tube, while the formation of germ tubes was observed in the spores grown in the RMPI medium. These results indicated that the M. purpureus pigment extract inhibited the germination of fungal spores. The spores treated with pigment extracts appeared red under the microscope due to the infiltration of the pigment through the destabilized membrane (Figure 2).

The physiological effects of pigment and amphotericin-B on treated mycelia were studied using a vital stain DiBAC, a fluorescent indicator of cell viability (Liao et al., 1999). The uptake of DiBAC stain was observed in the fungi treated with the pigment and amphotericin-B. The DiBAC stain entered the membrane-compromised cells and exhibited fluorescence (Figure 3). While the untreated mycelia grown in the RPMI medium showed no fluorescence (Figure 3A). These results indicated that amphotericin-B and pigment affected the membrane potential of the fungi. Extensive uptake of DiBAC stain indicated fungicidal activity of the M. purpureus pigment extracts. It was observed that amphotericin-B inhibited the fungi at 195 μg/ml and exerted fungicidal activity at 390 μg/ml, while the MIC was in the range of 780–1563 μg/ml and the MFC of the pigment extracts was 3,126 μg/ml. These results revealed that M. purpureus pigment extracts possess antifungal activity against the mycelia of P. expansum, MTCC 4900, R. stolinfer MTCC 10595, and A. niger MTCC 8652.

The pigment extracts were subjected to thin-layer chromatography (TLC) to observe the pigment molecules in the extracts. The TLC results revealed the presence of orange pigment prominently in ethyl acetate and ethanol extracts. While the orange pigment spot in the hexane extract was not observed clearly (Supplementary Figure 2). The pigment extracts subjected to the HPLC analysis showed two prominent peaks in hexane and ethyl acetate extracts. While three prominent peaks were observed in the ethanol extract. The pigments extracted in all the solvents have shown a common peak at a 12-min retention time (Figure 4). The UV–visible spectra analysis of this peak (Rt 12 min) has shown peaks at 248, 285, and 472 nm (Figure 4 inset).